Transfection of eukaryotic cells has become an increasingly important technique for the study and development of gene therapy. Advances in gene therapy depend in large part upon the development of delivery systems capable of efficiently introducing DNA into a target cell. A number of methods have been developed for the stable or transient expression of heterologous genes in cultured cell types. These include transduction techniques which use a carrier molecule or virus.
Most gene therapy strategies have relied on transduction by transgene insertion into retroviral or DNA virus vectors (Dimmock, N.J., "Initial stages in infection with animal viruses," J. Gen. Virol: (1982) 59:1-22; Duc-Nguyen, H., "Enhancing effect of diethylaminoethyl dextran on the focus forming titer of a murine sarcoma virus (Harvey strain)," J. Virol. (1968) 2:643-644). However, adenovirus and other DNA viral vectors can produce infectious sequelae, can be immunogenic after repeated administrations, and can only package a limited amount of insert DNA.
Of the viral vector systems, the recombinant adeno-associated viral (AAV) transduction system has proven to be one of the most efficient vector systems for stably and efficiently carrying genes into a variety of mammalian cell types (Lebkowski, J. S., et al., "Adeno-associated virus: A vector system for efficient introduction and integration of DNA into a variety of mammalian cell types," Mol. Cell. Biol. (1988) 8:3988-3996). It has been well-documented that AAV DNA integrates into cellular DNA as one to several tandem copies joined to cellular DNA through inverted terminal repeats (ITRs) of the viral DNA, and that the physical structure of integrated AAV genomes suggest that viral insertions usually appear as multiple copies with a tandem head to tail orientation via the AAV terminal repeats (Kotin, R. M., et al., "Site-specific integration of adeno-associated virus," Proc. Natl. Acad. Sci. (1990) 87:2211-2215; Samulski, R. J., et al., "Targeted integration of adeno-associated virus (AAV) into human chromosome 19," EMBO J. (1991) 10:3941-3950; Ashktorab, H. and A. Srivastara, "Identification of nuclear proteins that specifically interact with adeno-associated virus type 2 inverted terminal repeat hairpin DNA," J. Virol.(1989) 63:3034-3039; Im, D. S., and Muzyczka, N., "Factors that bind to adeno-associated virus terminal repeats," J. Virol.(1989) 63:3095-4104; Snyder, R. O., et al., "Evidence for covalent attachment of the adeno-associated virus (AAV) rep protein to the ends of the AAV genome," J. Virol.(1990) 64:6204-6213). Thus, the AAV terminal repeats (ITRs) are an essential part of the AAV transduction system.
Although recombinant adeno-associated viral (AAV) vectors differ from adenoviral vectors, the transgene DNA size limitation and packaging properties are the same as with any other DNA viral vectors.
AAV is a linear single stranded DNA parvovirus, and requires co-infection by a second unrelated virus in order to achieve productive infection. AAV carries two sets of functional genes: rep genes, which are necessary for viral replication, and structural capsid protein genes (Hermonat, P. L., et al., "Genetics of adeno-associated virus: Isolation and preliminary characterization of adeno-associated type 2 mutants," J. Virol.(1984) 51:329-339; Tratschin, J. D., et al., "Genetic analysis of adeno-associated virus: Properties of deletion mutants constructed in vivo and evidence for an adeno-associated virus replication function," J. Virol.(1984) 51:611-619). The rep and capsid genes of AAV can be replaced by a desired DNA fragment to generate AAV plasmid DNA. Transcomplementation of rep and capsid genes are required to create a recombinant virus stock. Upon transduction using such virus stock, the recombinant virus uncoats in the nucleus and integrates into the host genome by its molecular ends (Kotin, R. M., et al., "Site-specific integration of adeno-associated virus," Proc. Natl. Acad. Sci.(1990) 87:2211-2215; Samulski, R. J., et al., "Targeted integration of adeno-associated virus (AAV) into human chromosome 19," EMBO J.(1991) 10:3941-3950).
Although extensive progress has been made, transduction techniques suffer from variable efficiency, significant concern about possible recombination with endogenous virus, cellular toxicity and immunologic host response reactions. Thus, there is a need for non-viral DNA transfection procedures.
Liposomes have been used to encapsulate and deliver a variety of materials to cells, including nucleic acids and viral particles (regarding nucleic acids, see: Dimmock, N.J., "Initial stages in infection with animal viruses," J. Gen. Virol:(1982) 59:1-22; Duc-Nguyen, H., "Enhancing effect of diethylaminoethyl dextran on the focus forming titer of a murine sarcoma virus (Harvey strain)," J. Virol.(1968) 2:643-644); regarding viral particles, see: Felgner, P. L., et al., "A highly efficient, lipid-mediated DNA transfection procedure," Proc. Natl. Acad. Sci. USA(1987) 84:7413-7417; Faller, D. V. and D. Baltimore, "Liposome encapsulation of retrovirus allows efficient superinfection of resistant cell lines," J. Virol.(1984) 49:269-272; Wilson, T., et al., "Biological properties of poliovirus encapsulated in lipid vesicles: Antibody resistance and infectivity in virus resistant cells," Proc. Natl. Acad. Sci. USA(1977) 74:3471-3475).
Preformed liposomes that contain synthetic cationic lipids have been shown to form very stable complexes with polyanionic DNA (Felgner, P. L., et al., "A highly efficient, lipid-mediated DNA transfection procedure," Proc. Natl. Acad. Sci. USA(1987) 84:7413-7417; Rose, J. K., et al., "A new cationic liposome reagent mediating nearly quantitative transfection of animal cells," Biotechniques(1991) 10:520-525). Cationic liposomes, liposomes comprising some cationic lipid, that contained a membrane fusion-promoting lipid distearoyl-phosphatidyl-ethanolamine (DSPE) have efficiently transferred heterologous genes into eukaryotic cells (Felgner, P. L., et al., "A highly efficient, lipid-mediated DNA transfection procedure," Proc. Natl. Acad. Sci. USA(1987) 84:7413-7417; Rose, J. K., et al., "A new cationic liposome reagent mediating nearly quantitative transfection of animal cells," Biotechniques(1991) 10:520-525). Cationic liposomes can mediate high level cellular expression of transgenes, mRNA (Malone, R., et al., "Cationic liposome mediated RNA transfection," Proc. Natl. Acad. Sci. USA(1989) 86:6077-6081), or transcription factors (Debs, R., et al., "Regulation of gene expression in vivo by liposome-based delivery of a purified transcription factor," J. Biol. Chem.(1990) 265:10189-10192), by delivering them into a wide variety of cultured cell lines noted in these citations.
Ecotropic and amphotropic packaged retroviral vectors have been shown to infect cultured cells in the presence of cationic liposomes, such as Lipofectin (BRL, Gaithersburg, Md.), and in the absence of specific receptors (Innes, C. L., et al., "Cationic liposomes (Lipofectin) mediate retroviral infection in the absence of specific receptors," J. Virol.(1990) 64:957-961).
Overall, cationic liposomes have been shown to spontaneously complex with plasmid DNA or RNA in solution; the liposome comprising nucleic acids then facilitates fusion with cells in culture, resulting in the efficient transfer of nucleic acids to a wide variety of eukaryotic cell types. Liposome vectors are not subject to the DNA size and packaging properties that limit recombinant AAV vectors and adenoviral vectors. Thus, viral infection has been enhanced by coating virus with cationic liposomes and efficiently delivering the virus into cells.
Even though non-viral techniques have overcome some of the problems of the viral systems, there remains a need for improved transfection efficiency in non-viral systems (Hug, P. and R. G. Sleight, "Liposomes for the transformation of eukaryotic cells," Biochem. Biophys. Acta(1991) 1097:1-22; Felgner, P. L., et al., "A highly efficient, lipid-mediated DNA transfection procedure," Proc. Natl. Acad. Sci. USA(1987) 84:7413-7417). To a certain extent, improved efficiency is attained by the use of promoter enhancer elements in the plasmid DNA constructs (Philip, R., et al., "In vivo gene delivery: Efficient transfection of T lymphocytes in adult mice," J. Biol. Chem.(1993) 268:16087-16090).